Project Summary/Abstract Virus components such as the HIV-1 capsid and virus-encoded RNA can trigger powerful innate defense mechanisms when sensed by the host cell. Tripartite motif (TRIM) proteins are ubiquitin E3 ligases that perform important roles in sensing these virus components: TRIM5a proteins are established to recognize the incoming capsids of HIV-1 and retroviruses, and TRIM25 has an important but still enigmatic role in sensing viral RNA. The principal goals of this R01 project are to elucidate the molecular mechanisms of these sensors. In Aim 1, we propose to: (1) elucidate the molecular details of how TRIM5a recognizes HIV-1 and retroviral capsids through an unprecedented mechanism of lattice-to-lattice pattern recognition, (2) understand how multiple different protein-protein interactions in the TRIM5a/capsid complex cooperate to determine restriction susceptibility, and (3) develop novel biochemical reconstitution methods to recapitulate and analyze TRIM5a- mediated restriction in vitro. In Aim 2, we propose to: (1) elucidate the molecular details of how TRIM25 recognizes viral RNA, and (2) understand how this activity is coordinated with ubiquitination in order to enable recognition of both viral genomes and virally-encoded messenger RNA. Insights from these studies are likely to suggest novel ways to fight HIV-1 and other viral infections.